It must be noted that as used herein and in the appended claims, the singular forms “a” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a cell” or “the cell” includes a plurality (“cells” or “the cells”), and so forth. Moreover, the word “or” can either be exclusive in nature (i.e., either A or B, but not A and B together), or inclusive in nature (A or B, including A alone, B alone, but also A and B together). One of skill in the art will realize which interpretation is the most appropriate unless it is detailed by reference in the text as “either A or B” (exclusive “or”) or “and/or” (inclusive “or”).
The inventor can be contacted at hildinger@gmx.net.
A portion of the disclosure of this patent document contains material which is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure as it appears in the Patent and Trademark Office file or records, but otherwise reserves all copyright rights whatsoever.
(1) Field of the Invention
This invention relates to AAV-mediated gene transfer in general and to AAV-mediated transfer of (recombinant) AAV genomes larger than 5.7 kb, 6 kb, 6.5 kb, 7 kb, 7.5 kb, 7.8 kb or 8 kb in particular. In that respect, the present invention will find use in gene therapy in general, and particularly in gene therapy applications where large recombinant genomes will have to be transferred. In prefrerred embodiments, the AAV vectors of the present invention will comprise recombinant AAV genomes, where said recombinant AAV genomes comprise transgene expression cassettes larger than 6 kb, 6.5 kb, 7 kb or 7.5 kb, and where said transgene expression cassettes comprise a nucleotide sequence encoding the ABCR protein (as referenced in SEQ ID NO: 2), the Factor VIII protein (as referenced in SEQ ID NO: 4), a B-deleted Factor VIII protein (as referenced in SEQ ID NO: 6) or a minidystrophin protein (as referenced in SEQ ID NO: 8).
The size of the recombinant AAV genome of the present invention depends on the application. Examples for the need to transfer recombinant AAV genomes larger than 5.7 kb are:    The transfer of coding sequences close to or larger than 5.7 kb such as the full-length ABCA4 coding sequence (as referenced in SEQ ID NO: 1), the Factor VIII coding sequence (as referenced in SEQ ID NO: 3), or a minidystrophin coding sequence (as referenced in SEQ ID NO: 7).    Further examples are long coding sequences which—in combination with regulatory elements and indispensable AAV cis elements—exceed at least 5.7 kb or can exceed at least 5.7 kb, such as the CFTR coding sequence (as referenced in SEQ ID NO: 32), the B-deleted Factor VIII coding sequence (as referenced in SEQ ID NO: 5), the Usherin-2a coding sequence (as referenced by SEQ ID NO: 34).    Other examples are self-complementary AAV vectors where the transgene expression cassette including AAV cis elements exceeds at least 2.85 kb, at least 3 kb, at least 3.25 kb, at least 3.5 kb, at least 3.75 kb or at least 3.9 kb, or at least 4.0 kb, such as self-complementary AAV vectors harboring the PDE 6b coding sequence (referenced by SEQ ID NO: 58) in combination with regulatory sequences and/or AAV cis elements, where said regulatory sequences and/or AAV cis elements exceed 300 nucleotides.
Thus, the present invention will find use in medical applications in the context of gene therapy to treat diseases such as                Stargardt Disease, by transducing affected cells with an AAV vector harboring an ABCA4 expression cassette;        Hemophilia A, by transducing mammalian cells with an AAV vector harboring a Factor VIII expression cassette, or by transducing mammalian cells with an AAV vector harboring a B-deleted Factor VIII expression cassette;        Duchenne Muscular Dystrophy (DMD), by transducing affected cells with an AAV vector harboring a minidystrophin expression cassette;        Cystic fibrosis (CF), by transducing affected cells with an AAV vector of the present invention harboring a CFTR expression cassette as well as additional elements, where those additional elements exceed ˜1.3 kb.        
Those diseases are caused by mutations in genes whose coding sequence exceed (apart from Cystic Fibrosis) 5.5 kb, which—according to prior art—would exceed in combination with regulatory elements and AAV cis elements the effective packaging capacity of AAV vectors. Yet, the present invention is not limited to the treatment of the diseases listed above, but generally appicable to the AAV-mediated transfer of recombinant AAV genomes larger than 5.7 kb, 6 kb, 6.5 kb, 7 kb, 7.5 kb, 7.8 kb or 8 kb. For example, the AAV-mediated transfer of a 3 kb coding sequence in combination with a 3 kb promoter sequence as part of a transgene expression cassette would still fall within the scope of the present invention.
(2) Description of Related Art
The inventor would like to call particular attention to section (b) of “Description of Related Art”, where the effective AAV packaging capacity as well as the AAV packaging limit are discussed.
(a) Adeno-Associated Viral Vectors
Adeno-associated virus (AAV) is a small non-pathogenic virus of the parvoviridae family. AAV is distinct from the other members of this family by its dependence on a helper virus for replication. The approximately 4.7 kb genome of AAV consists of single stranded DNA of either plus or minus polarity. The ends of the genome are short inverted terminal repeats (ITRs) which can fold into hairpin structures and serve as the origin of viral DNA replication. Physically, the parvovirus virion is non-enveloped and its icosohedral capsid is approximately 20 nm in diameter. To date, at least 11 serologically distinct AAVs have been identified and isolated from humans or primates and are referred to as AAV serotypes 1-11.
The genome of AAV2 is 4,680 nucleotides in length and contains two open reading frames (ORFs): The left ORF encodes the non-structural Rep proteins, Rep40, Rep52, Rep68 and Rep78, which are involved in regulation of replication and transcription in addition to the production of single-stranded progeny genomes. Furthermore, two of the Rep proteins have been associated with the preferential integration of AAV2 genomes into a region of the q arm of human chromosome 19. Rep68/78 have also been shown to possess NTP binding activity as well as DNA and RNA helicase activities. The Rep proteins possess a nuclear localization signal as well as several potential phosphorylation sites. Mutation of one of these kinase sites resulted in a loss of replication activity. The ends of the genome are short inverted terminal repeats which have the potential to fold into T-shaped hairpin structures that serve as the origin of viral DNA replication. Within the ITR region two elements have been described which are central to the function of the ITR, a GAGC repeat motif and the terminal resolution site (trs). The repeat motif has been shown to bind Rep when the ITR is in either a linear or hairpin conformation. This binding serves to position Rep68/78 for cleavage at the trs which occurs in a site- and strand-specific manner. In addition to their role in replication, these two elements appear to be central to viral integration. Contained within the chromosome 19 integration locus is a Rep binding site with an adjacent trs. These elements have been shown to be functional and necessary for locus specific integration.
The right ORF of AAV2 encodes related capsid proteins referred to as VP1, 2 and 3. These capsid proteins form the icosahedral, non-enveloped virion particle of ˜20 nm diameter. VP1, 2 and 3 are found in a ratio of 1:1:10. The capsid proteins differ from each other by the use of alternative splicing and an unusual start codon. Deletion analysis has shown that removal or alteration of VP1, which is translated from an alternatively spliced message, results in a reduced yield of infectious particles. Mutations within the VP3 coding region result in the failure to produce any single-stranded progeny DNA or infectious particles.
The findings described in the context of AAV2 are generally applicable to other AAV serotypes as well.
The following features of AAV have made it an attractive vector for gene transfer: AAV vectors possess a broad host range, transduce both dividing and non dividing cells in vitro and in vivo and maintain high levels of expression of the transduced genes in the absence of a significant immune response to the transgene product in general. Moreover, as wild-type AAV is non-pathogenic, AAV vector particles are assumed to be non-pathogenic as well (in contrast to adenoviral vectors). Viral particles are heat stable, resistant to solvents, detergents, changes in pH and temperature. The ITRs have been shown to be the only cis elements required for replication and packaging and may contain some promoter activities. Thus, no viral genes are encoded by AAV vectors.
Vectors based on adeno-associated virus (AAV) emerged as those preferred for achieving truly stable transduction following in vivo administration. The recent isolation and characterization of several new AAV serotypes provides new opportunities for vector development. For example, Chiorini and colleagues created replication defective versions of AAV serotype 5 (AAV5) for gene transfer. Transduction efficiency was substantially improved with AAV5-based vectors when compared with those based on AAV2 in several applications, including those involving muscle and lung. Another improvement in the art was the creation of hybrid vectors based on AAV2 inverted terminal repeats (ITRs) produced with AAV2 rep and AAV5 cap. The resulting defective vector packages an AAV2 genome in an AAV5 capsid. The transduction efficiency of the AAV2/5 hybrid is superior to that of AAV2 in lung, muscle, and retina. A further advantage of AAV vectors based on serotype 5 capsids is that humans do not harbor antibodies capable of interfering with AAV5 transduction. There is also clinical experience using AAV vectors to safely transfer genes to human organs.
To summarize: AAV is a small non-enveloped icosahedral parvovirus with a 4.7-kb single-stranded DNA genome. AAV is a naturally replication-defective virus that depends on adenovirus (Ad) or herpes simplex virus gene products for replication. The absence of any detectable pathology from wild-type AAV infections coupled with its ability to remain latent promoted its development as a gene transfer vector. Recombinant vectors based on AAV are effective in long-term gene transfer to skeletal and cardiac muscle, liver, brain, and retina in the absence of an immune response even to non-self transgene products.
AAV vectors are designed in a fashion such that all viral genes are replaced by an expression cassette for the transgene, leaving intact the essential cis elements of the genome, the inverted terminal repeats (ITRs), DNA packaging signal, and the replication origin (Backlow, 1988). Replication and packaging of AAV vectors requires all AAV and Ad/ HSV helper functions to be provided in trans. Whereas wild-type AAV is capable of integrating in a site-specific manner into human chromosome 19, site-specific integration of recombinant AAV does not seem to occur to a significant extent (due to the lack of Rep Protein expression in AAV vectors). Moreover, the onset of gene expression is generally delayed by 2-4 weeks.
(b) Effective Packaging Capacity of AAV Virions
The inventor considers the following publications as essential prior art:                [A]: Dong et al.: “Quantitative analysis of the packaging capacity of recombinant adeno-associated virus.” in Hum Gene Ther. Nov. 10, 1996;7(17):2101-12.        [B]: Hermonat et al.: “The packaging capacity of adeno-associated virus (AAV) and the potential for wild-type-plus AAV gene therapy vector” in FEBS Lett. Apr. 21, 1997;407(1):78-84.        [C]: Ostedgaard et al.: “A shortened adeno-associated virus expression cassette for CFTR gene transfer to cystic fibrosis airway epithelia.” in Proc Natl Acad Sci USA. Feb. 22, 2005; 102(8):2952-7.        [D]: Flotte et al.: “Expression of the cystic fibrosis transmembrane conductance regulator from a novel adeno-associated virus promoter.”J Biol Chem. Feb. 15, 1993;268(5):3781-90.        [E]: Zhang et al.: “Efficient expression of CFTR function with adeno-associated virus vectors that carry shortened CFTR genes.” Proc Natl Acad Sci USA. Aug. 18, 1998;95(17):10158-63.        
The 1996 publication of Dong et al. [A] teaches an effective packaging capacity for AAV vectors of 4.1 to 4.9 kb and a packaging limit of 5.2 kb. I quote: “Our studies showed that the optimal size of AAV vector is between 4.1 and 4.9 kb. Although AAV can package a vector larger than its genome size, up to 5.2 kb, the packaging efficiencies in this large size range were sharply reduced.”
Similarly, the 1997 publication of Hermonat et al. [B] teaches an effective packaging capacity for AAV up to 119% of wild-type, or 5.6 kb. I quote: “These data indicate that the maximum effective packaging capacity of AAV is approximately 900 bp larger than wild type, or 119% . . . These data suggest that therapy vectors carrying a foreign gene of 900 bp or less can be generated from AAV.”
Another prior art publication of February 2005 by Ostedgaard et al. [C] states: “The 6,065-bp total length exceeds the packaging capacity of AAV (refs. 14-16 and unpublished observations). Substituting the recently developed shortened CFTR transgene, CFTRΔR (4,287 bp) (24), reduced the cassette length to 5,902 bp. However, this still exceeds the packing limits.” This publication further claims: “A limitation of AAV vectors is the relatively small AAV genome. Studies testing the insert size suggest that 4,100-4,900 bp is the optimal genome size for packaging (14). Other studies and our own unpublished data also suggest that packaging becomes very inefficient whenever insert sizes exceed 4,900-5,000 bp (15, 16). This poses a problem for genes with large coding sequences . . . ” The authors of that publication constructed then a vector smaller than 5 kb in size.
Contrary to those prior-art publications, the inventor discovered that the effective packaging capacity of AAV is larger than 5.7 kb, larger than 6 kb, larger than 6.5 kb, larger than 7 kb, larger than 7.5 kb, larger than 7.8 kb and larger than 8 kb. Moreover, the inventor discovered that the AAV packaging limit is larger than 5.7 kb, larger than 6 kb, larger than 6.5 kb, larger than 7 kb, larger than 7.5 kb, larger than 7.8 kb and larger than 8 kb. In detail, the inventor discovered that the effective packaging capacity and the packaging limit vary as a function of serotype: Whereas the effective packaging capacity and packaging limit for AAV2 is in the range of published prior art data, this is not true for other serotypes such as recombinant AAV vectors comprising an AAV5 capsid or an AAV7 capsid. The capsid of AAV5 and the capsid of AAV7 seem to be able to accommodate larger genomes. Particularly AAV5 packages large genomes even beyond 6.5 kb and up to 8 kb with high efficacy.
(c) Stargardt Disease
Stargardt disease, also known as fundus flavimaculatus, is the most common form of inherited juvenile macular degeneration. It is characterized by a reduction of central vision with a preservation of peripheral (side) vision. Stargardt disease is usually diagnosed in individuals under the age of 20 when decreased central vision is first noticed. On examination, the retina of an affected individual shows a macular lesion surrounded by yellow-white flecks, or spots, with irregular shapes. The retina consists of layers of light-sensing cells that line the inner back wall of the eye and are important in normal vision. The macula is found in the center of the retina and is responsible for the fine, detailed central vision used in reading and color vision.
The progression of visual loss is variable. One study of 95 individuals with Stargardt disease showed that once a visual acuity of 20/40 was reached, there was often rapid progression of additional visual loss until acuity was reduced to 20/200 (legal blindness). By age 50, approximately 50 percent of all those studied had visual acuities of 20/200 or worse. Eventually, almost all individuals with Stargardt disease are expected to have visual acuities in the range of 20/200 to 20/400. The reduced visual acuity due to Stargardt disease cannot be corrected with prescription eyeglasses or contact lenses. In late stages of the disease, there may also be noticeable impairment of color vision.
Stargardt disease is almost always inherited as an autosomal recessive disorder. It is inherited when both parents, called carriers, have one gene for the disease paired with one normal gene. Carriers are unaffected because they have only one copy of the gene. The gene responsible for Stargardt disease has been identified as the ABCA4 gene, which encodes the ABCR protein (referenced by SEQ ID NO: 2). ABCR stands for “ATP-binding cassette transporter—retinal”.
The ABCR protein plays an important role in the visual cycle: All-trans retinal, which is released into the disc lumen of the photoreceptor cells, reacts with phosphatidyl ethanolamine (PE) to N-retinylidene-PE, which is subsequently transported into the cytosol by the function of the ABCR. Thus, ABCR is the rate keeper of retinal transport in the visual cycle. If ABCR function is lost, N-retinylidene-PE accumulates in the disc lumen. Once the discs are phagocytosed by Retinal Pigment Epithelium (RPE) cells, excessive N-retinylidene-PE is transformed into N-retinylidine-N-retinylethanolamine (A2-E), which is a major component of lipofuscin. Accumulation of lipofuscin leads to RPE cell apoptosis. Thus, mutations in the ABCR gene produce a dysfunctional protein that cannot perform its transport function. As a result, photoreceptor cells degenerate and vision loss occurs. The most common mutations, accounting for 10% of all cases of autosomal recessive Stargardt Disease, are G1961E, G863A, ΔG863, and A1038V.
(d) Hemophilia A
Hemophilia A is a hereditary blood coagulation (clotting) disorder. It is caused by a deficient activity of plasma protein factor VIII (referenced by SEQ ID NO: 4), which affects the clotting property of blood. Hemophilia A is the most common of blood coagulation disorder. The disorder is caused by an inherited X-linked recessive trait, with the defective gene located on the X chromosome. Thus, the disorder occurs primarily in males. Females carry two copies of the X chromosome, so if the factor VIII gene on one chromosome is defective, the gene on the other chromosome can compensate. Males, however, carry only one X chromosome, so if the factor VIII gene on that chromosome is defective, they will have the disease.
The human Factor VIII cDNA (FVIII cDNA) has been cloned. FVIII is synthesized as a 2351 amino acid residue, single chain precursor composed of a 19 amino acid signal peptide and six distinct domains. The domains are arranged in the order, A1-A2-B-A3-C1-C2. An A domain contains about 330 amino acids and is present in three copies. A C domain contains about 150 amino acids and is present in two copies. The B domain contains about 909 amino acids and is extremely rich in potential N-linked glycosylation sites. The translation product of the FVIII gene first is cleaved between the B domain and the A3 domain. Then, the B domain is proteolysed at multiple sites leaving FVIII as a divalent metal ion-linked complex consisting of the heavy chain (H chain) of 90-200 kDa and the light chain (L chain) of 80 kDa. The minimal functional unit of FVIII is the heterodimer consisting of the 90 kDa H chain and the 80 kDa L chain. Thus, the B domain is dispensable for procoagulant activity. Circulating FVIII in blood is associated with the von Willebrand factor (vWF) which is a large multimeric, multifunctional product. Expression of full-length FVIII cDNA in mammalian cells was reported by several groups, but the levels of expression were very low and insufficient for economical production of recombinant FVIII (rFVIII). To improve expression efficiency, modified FVIII cDNA's lacking most of the B domain were made and the resulting products were shown to retain functional activities of FVIII.
The severity of symptoms can vary with this disease, and the severe forms become apparent early on. Bleeding is the hallmark of the disease and sometimes, though not always, occurs if an infant is circumcised. Additional bleeding manifestations make their appearance when the infant becomes mobile.
Mild cases may go unnoticed until later in life when they occur in response to surgery or trauma. Internal bleeding may happen anywhere, and bleeding into joints is common. Risk factors are a family history of bleeding and being male. Hemophilia A occurs in about 1 out of 5,000 men. Symptoms are bruising, spontaneous bleeding, bleeding into joints and associated pain and swelling, gastrointestinal tract and urinary tract hemorrhage, blood in the urine or stool, prolonged bleeding from cuts, tooth extraction, and surgery.
Many blood clotting tests are performed if the person tested is the first one in the family to have a bleeding disorder. Once the defect has been identified, other family members will need less testing to diagnose the disorder. Tests include prolonged PTT, normal prothrombin time, normal bleeding time, normal fibrinogen level, low serum factor VIII activity.
Standard treatment is infusion of factor VIII concentrates to replace the defective clotting factor. The amount infused depends upon the severity of bleeding, the site of the bleeding, and the size of the patient. Mild hemophilia may be treated with infusion of cryoprecipitate or desmopressin (DDAVP), which causes release of factor VIII that is stored within the body on the lining of blood vessels. To prevent a bleeding crisis, people with hemophilia and their families can be taught to administer factor VIII concentrates at home at the first signs of bleeding. People with severe forms of the disease may need regular prophylactic infusions. Depending on the severity of the disease, DDAVP or factor VIII concentrate may be given prior to dental extractions and surgery to prevent bleeding.
With treatment, the outcome is good. Most people with hemophilia are able to lead relatively normal lives. A small percentage of people with hemophilia will develop inhibitors of factor VIII, and may die from loss of blood.
Complications include chronic joint deformities, caused by recurrent bleeding into the joint, and should be managed by an orthopedic specialist. These problems sometimes require joint replacement. Recurrent transfusions may increase the risk of contracting HIV and hepatitis, especially prior to 1985 when blood screening procedures were improved for detecting the HIV virus. However, new heat processing treatment makes factor VIII material free of the HIV virus and thus safe for use. Intracerebral hemorrhage is another possible complication (see deep intracerebral hemorrhage, lobar intracerebral hemorrhage).
(e) Duchenne Muscular Dystrophy
Duchenne muscular dystrophy is an inherited disorder characterized by rapidly progressive muscle weakness which starts in the legs and pelvis and later affects the whole body. It is caused by a defective gene, the dystrophin gene, but it often occurs in people from families without a known family history of the condition. It is marked by progressive loss of muscle function, which begins in the lower limbs. The cause of the muscle impairment is an abnormal gene for dystrophin (a protein in the muscles).
Duchenne muscular dystrophy is inherited in an X-linked recessive pattern. Because women have two X chromosomes, if one contains a normal copy of the gene, that gene will make enough of the protein to prevent symptoms. But boys have an X chromosome from their mother and a Y from father, so if the X chromosome is defective, there is no second X to make up for it and they will develop the disease.
Symptoms usually appear before age 6 and may appear as early as infancy. There is progressive muscle weakness of the legs and pelvis, which is associated with a loss of muscle mass (wasting). Muscle weakness also occurs in the arms, neck, and other areas, but not as severely or as early as in the lower half of the body. Calf muscles initially enlarge—the enlarged muscle tissue is eventually replaced by fat and connective tissue (pseudohypertrophy). Muscle contractures occur in the legs, rendering the muscles unusable because the muscle fibers shorten and fibrosis occurs in connective tissue.
Symptoms usually appear in boys aged 1-6. By age 10, braces may be required for walking, and by age 12, most patients are confined to a wheelchair. Bones develop abnormally, causing skeletal deformities of the spine and other areas. Muscular weakness and skeletal deformities contribute to frequent breathing disorders. Cardiomyopathy occurs in almost all cases. Intellectual impairment may occur, but it is not inevitable and does not worsen as the disorder progresses.
Duchenne muscular dystrophy occurs in approximately 2 out of 10,000 people. Because this is an inherited disorder, risks include a family history of Duchenne muscular dystrophy. In contrast, Becker muscular dystrophy is a form that progresses much more slowly.
Symptoms include muscle weakness, rapidly progressive, frequent falls, difficulty with motor skills (running, hopping, jumping), progressive difficulty walking, ability to walk may be lost by age 12, fatigue, intellectual retardation (possible), skeletal deformities, chest and back (scoliosis), muscle deformities, contractures of heels and legs, pseudohypertrophy of calf muscles. Muscle wasting (atrophy) begins in the legs and pelvis, then progresses to the muscles of the shoulders and neck, followed by loss of arm muscles and respiratory muscles. Calf muscle enlargement (pseudohypertrophy) is quite obvious.
Cardiomyopathy is commonly present, but signs of congestive heart failure or arrhythmias (irregular heartbeats) are rare. Respiratory disorders are common during the later stages, including pneumonia and aspiration of food or fluid into the lungs.
As far as diagnosis is concerned, a serum CPK is highly elevated. A neurologic exam demonstrates weaness and lack of coordination or balance. An EMG (electromyography) shows that weakness is caused by destruction of muscle tissue rather than nerve damage. A muscle biopsy confirms the diagnosis.
There is no known cure for Duchenne muscular dystrophy. Treatment is aimed at control of symptoms to maximize the quality of life. Gene therapy may become available in the future. Activity is encouraged. Inactivity (such as bedrest) can worsen the muscle disease. Physical therapy may be helpful to maintain muscle strength and function. Orthopedic appliances (such as braces and wheelchairs) may improve mobility and the ability for self-care. The stress of illness can often be helped by joining a support group where members share common experiences and problems. See muscular dystrophy—support group. The Muscular Dystrophy Association is an excellent source of information on this disease.
Duchenne muscular dystrophy results in rapidly progressive disability. Death usually occurs by age 25, typically from respiratory (lung) disorders. Complications include deformities, permanent, progressive disability, decreased mobility, decreased ability for self-care, mental impairment (varies, usually minimal), pneumonia or other respiratory infections, respiratory failure, cardiomyopathy, congestive heart failure (rare), heart arrhythmias (rare).